HIV-1 protein Nef is a multifunctional protein involved in regulation of viral infectivity and responsible for many pathogenic effects of HIV infection, including downregulation of CD4, MHC I and ABCA1. The effect of Nef on host cell proteins has been ascribed to the ability of Nef to function as a bridge between the host cell transport proteins and the target protein, thus promoting transport and degradation of the latter by the cellular lysosomal or proteasomal machinery. In our preliminary experiments, we identified a novel interaction partner of Nef, the endoplasmic reticulum (ER) chaperone calnexin. Calnexin is an integral ER transmembrane protein the main function of which is to assist protein folding and perform quality control during maturation of N-linked glycoproteins. We found that Nef disrupts interaction between calnexin and cholesterol transporter ABCA1, resulting in release from ER of non-functional ABCA1 and inhibition of cholesterol efflux. However, this effect of Nef was selective, as calnexin interaction with another glycoprotein, HIV-1 gp160, was not disrupted. The mechanism responsible for this selectivity is unknown, but Nef is known to affect localization of proteins to which it binds. Based on these preliminary studies, we hypothesize that Nef interaction with calnexin alters calnexin localization promoting its interaction with gp16 at the expense of a certain repertoire of host cell proteins whose maturation and function are impaired. This hypothesis will be tested in Aim 1 of the proposal. In Aim 2, we will use bioinformatics, molecular modeling and mutagenesis of Nef and calnexin, to identify the motifs and domains responsible for the interaction between these proteins and will test available Nef-targeting drugs and new compounds identified by virtual screening for the ability to interfere with this interaction. The bioinformatics studies will be performed by our Russian collaborator, Dr. Adzhubei. In Aim 3, we will characterize functional consequences of Nef effects on calnexin for the virus, cell and host organism. Using mass-spectrometry, we will assess glycosylation of gp160 and ABCA1 in the presence and absence of Nef. We will also test drugs targeting Nef-calnexin interaction in Nef-transgenic mice available in the laboratory of another Russian collaborator, Dr. Nedospasov. These studies will characterize novel mechanism behind the effects of Nef on host cell and viral proteins, may provide an explanation for the stimulatory effect of Nef on HIV infectivity, will identify new anti-HIV agents, and may inform anti-HIV vaccine design efforts.